Method for electrochemiluminescent labeling of nucleic acid, use in detection, and compositions therefor

ABSTRACT

Method for immobilizing nucleic acids by hybridizing the nucleic acid with a capture probe. The capture probe is protected against enzymatic extension and/or enzymatic degradation of the formed hybrid.

This is a division of application Ser. No. 08/257,778 filed Jun. 9,1994; now U.S. Pat. No. 5,639,609.

Subject matter of the invention are methods for immobilizing nucleicacids, methods for detecting nucleic acids making use of thisimmobilization, reagents for implementing this method and correspondingreagent kits.

When detecting nucleic acids, a distinction is made between homogeneousand heterogeneous assay methods. In homogeneous methods, the detectionprobes and the nucleic acids to be detected are assayed in solution,whereas in heterogeneous methods the nucleic acid to be detected isimmobilized. This immobilization is achieved either by directlyattaching the nucleic acid to a solid phase via covalent binding or bycoupling the nucleic acid to one partner of an affinity pair whilebinding the remaining partner of this pair to a solid phase.

Coupling can also be achieved by immobilizing the nucleic acid byhybridizing it to a so-called capture probe which is bound to a solidphase and at least partly complementary to the nucleic acid to bedetected. The capture probe is or can be bound to a solid phase in anydesired manner. An advantage of heterogeneous methods is that labelledreagent components can be easily separated from the nucleic acid to bedetected.

Examples for methods where the nucleic acids to be detected areimmobilized via capture probes can be found in EP-A-0 079 139 whichdescribes direct binding and in EP-A-0 192 168 which describes bindingvia an affinity pair. A characteristic feature of the assay methodsdescribed therein is the hybridization of the nucleic acid to bedetected with a capture probe. After removing excess detection probe,the so formed hybrid is then detected at the solid phases via alabelling group of the capture probe.

With the introduction of target-specific amplification processes tonucleic diagnostics, it was possible to produce large amounts ofotherwise rarely occurring nucleic acids. A method of this kind where adigoxigenin label is incorporated into the amplified nucleic acid, isdescribed in WO 92/06216.

PCT/US 92/01735 discloses a method for preparing 2'-substituted nucleicacids and their resistance to degradation by restriction enzymes.Further, it is also known that when nucleic acids are sequenced,3'-deoxynucleotides truncate chains and inhibit the extension of nucleicacids in 3'-direction.

It is an object of the present invention to provide a particularlysimple and efficient method of immobilization.

Subject matter of the invention is a method for immobilizing nucleicacids by hybridizing the nucleic acid with a capture probe whereby thecapture probe is protected against enzymatic extension and/or enzymaticbreak-down of the formed DNA/RNA-hybrid.

Other subject matters of the invention are a method for detecting anucleic acid making use of the immobilization according to this methodand reagents suitable for this purpose.

A capture probe is a nucleic acid which can hybridize with anothernucleic acid or which is or can be immobilized. Immobilization is aprocedure whereby a nucleic acid is bound to a solid phase. This caneither be a direct binding (covalent binding of the capture probe to thesurface of the solid phase) or an indirect binding (ionic, adsorptive,or biospecific binding). In the invention, preferred nucleic acid arenucleic acids which are labelled with a partner of a biospecific pairwhile allowing immobilization. Biospecific pairs are such combinationsas antigen/antibody, hapten/antibody, vitamin/receptor,hormone/receptor, and sugar/lectin. Preferred combinations arebiotin/avidin, biotin/streptavidin, or hapten/antibody. So labellednucleic acids can be prepared, for example, by means of chemicaloligonucleotide synthesis using correspondingly labelled activemononucleoside derivatives or by means of enzymatic synthesis viaincorporation of correspondingly labelled mononucleoside triphosphatesor by chemically modifying fully synthesized nucleic acids.

Enzymatic extension is a process that occurs in living cells or influids deriving from such cells which still contain active enzymes. Theprocess is triggered by enzymes such as polymerases which extend nucleicacids. These extension reactions may significantly interfere with thedetermination of nucleic acids, particularly when corresponding enzymeshave been added to the sample to carry out the amplification process.

In order to suppress this extension reaction, the invention proposes toprotect the capture probe against extension. This can be achieved byattaching chemical residues which inhibit steric access of extensionenzymes to the 3'- or 5'-end. As these residues sometimes inhibit oreven completely prevent hybridization of the capture probe with theimmobilizing nucleic acid, it is preferred to replace the hydroxyl groupat which the extension would take place by an inactive group.

Such groups include, for example, hydrogen or C1-C3-alkyl groups, withhydrogen being preferred. At their ends, the so obtained capture probes,therefore, contain a 3'- or 5'-deoxyribonucleoside residue or a 2',3'-or 2',5'-dideoxyribonucleoside residue. The 3'-end of the capture probeis the preferred end to be protected.

Enzymatic break-down is also a common phenomenon in biological sampleliquids. Such a break-down is caused by endo- or exonucleases such asDNAses or RNAses. This also includes restriction enzymes. If, forexample, RNA is to be used as a nucleic acid to be immobilized, this RNAwould be subject to degradation by RNAseH as soon as a hybrid consistingof DNA as the capture probe and the RNA is formed. This meansimmobilization by means of unmodified DNA capture probes is veryinefficient or even impossible. The present invention attempts toeliminate this by modifying the capture probe with the result that theabove mentioned enzymes no longer recognize the hybrid of capture probeand nucleic acid to be detected as substrate.

The modification of sugar residues of the capture probes, e.g., at the2'-position, has proven to be quite efficient. A particularly preferredcombination for the immobilization of RNA in the presence of RNAseH isone where the 2'-hydroxyl group of deoxyribonucleotides is replaced byO-alkyl or O-alkylene groups. Particularly preferred combinations aredescribed in WO 91/15499.

Surprisingly, it is possible to protect nucleic acids effectivelyagainst enzymatic break-down of the RNA/DNA-hybrid and enzymaticextension by simultaneously employing the described measures. Thismodifications are selected such that their effects and actions do notnegatively affect one another.

Capture probes include oligo or polynucleotides which can also befurther modified. However, this modification must exclude thepossibility that hybridization is rendered impossible by base-pairingwith the nucleic acid to be immobilized.

Immobilized capture probes are nucleic acids which are bound to a solidphase. The binding can be achieved by forming covalent links orbiospecifically via two partners forming a biospecific link (e.g.hapten/antibody, vitamin/receptor, preferably biotin/streptavidin)whereby one of the partners is bound to the capture probe while theother is bound to the solid phase.

Capture probes which can be immobilized are nucleic acids which are notyet bound to a solid phase, but which can be turned into the abovementioned immobilized capture probes. To achieve this, the immobilizedcapture probes contain either a residue capable of chemically reactingwith the solid phase (e.g. residues which can be photoactivated) or apartner of a biospecific binding.

Possible solid phases include cuvettes, microtiterplates or particles(e.g. plastic-coated magnetic beads).

To achieve an immobilization of the nucleic acids in accordance with theinvention, the capture probe is brought into contact with a sample thatcontains the nucleic acid to be immobilized. Hybridization is carriedout under conditions which depend on the length of the nucleic acid usedand the capture probe. The expert is capable of determining theseconditions in a few experiments, e.g. by determining the meltingtemperature. Generally, however, these conditions will not differ fromthose for nucleic acids having the same (unmodified) nucleotide sequenceas the capture probe.

If the capture probe that is used is an already immobilized nucleicacid, hybridization of the capture probe with nucleic acid occurssimultaneously with the immobilization of the nucleic acid. If desire,the hybrid formed at the solid phase can now be separated from theliquid which contained the nucleic acid. Subsequently, the solid-phasebound hybrid can be washed and is available for further reactions, ifnecessary.

If the capture probe is one that can be immobilized, thehybrid-containing liquid is brought into contact with a solid phasecapable of immobilizing the capture probe. To achieve this, the solidphase preferably contains a binding partner of the group used toimmobilize the capture probe. As is the case with immobilized captureprobes, further processing steps may follow.

The nucleic acid to be immobilized can be one of any desired kind and ofany desired origin. These may be nucleic acids occurring in naturalsamples, e.g. body fluids or cultured samples, but also samples obtainedby processing natural samples. They can, hence, be modified orunmodified. The modifications can relate to the sequence, length and/orthe chemical structure of the nucleic acids. In a preferred case, thenucleic acids are copies of a nucleic acid or a nucleic acid sequencethat was originally contained in the sample. Moreover, preferred copiesare amplification products resulting from an in-vitro amplification of apart of the original nucleic acid. Particularly preferred amplificationproducts are those formed as templates by enzymatic extension of thenucleic acid at the original nucleic acid. Examples for such in-vitroamplifications are PCR, LCR or promoter-controlled amplifications.

In another preferred embodiment, the nucleic acids are modified byincorporating detectable groups. This incorporation can be implementedvia enzymatic or chemical reactions. Enzymatic reactions areparticularly preferred as they occur during amplification as describedabove. In this case, it has proven to be particularly expedient to usedetectable modified monocucleoside triphosphates for the extension. Inpreferred manner, all non-incorporated detectable groups are thenseparated. If the nucleic acid to be immobilized is modified such thatit can be detected, it can be detected at the solid phase in a knownmanner once it is immobilized. The detection step may, hence, follow theimmobilization process. Another subject matter of the invention is amethod for detection nucleic acid using the immobilization process ofthe invention followed by the detection of the immobilized hybrids. In aparticularly preferred manner, the detectable groups are determined atthe immobilized nucleic acid.

Detectable groups are directly or indirectly detectable groups. Directlydetectable groups include, for example, radioactive (hu 32P), colored,or fluorescent groups or metal atoms. Indirectly detectable groupsinclude, for example, immunological or enzymatically effective compoundssuch as antibodies, antigens, haptens, or enzymes, or enzymaticallyactive partial enzymes. They are detected in a subsequent reaction orsequence of reactions. Haptens are particularly preferred as theygenerally perform well with labelled nucleoside triphosphates assubstrates of polymerases and as the subsequent reaction with a labelledantibody to the hapten or the haptenized nucleoside can be readilyimplemented. Such nucleoside triphosphates are, for example,bromine-nucleoside triphosphate or digoxigenin, digoxin- orfluorescein-coupled nucleoside triphosphates. The steroids described inEP-A-0 324 474 and their detection have proven to be particularlysuitable. For details on their incorporation refer to EP-A-0 324 474.

In a first embodiment of the method of the invention, the nucleic acidto be detected is detected via a detection probe which is hybridized toit. This detection probe has a nucleotide sequence which can hybridizeto a segment of the nucleic acid to be detected, said segment beingdifferent from the one to which the capture probe hybridizes. The resultis a so-called sandwich hybrid consisting of capture probe, nucleic acidto be detected and detection probe. The hybridization sequence of thesethree nucleic acids can principally be freely selected. In a preferredmanner, sandwich formation occurs essentially simultaneously. In aparticularly preferred embodiment of the sandwich test, the captureprobe is added to the sample containing the nucleic acid to be detected.Subsequently, the nucleic acid to be detected or part thereof areamplified and then the hybrids formed under hybridization conditions aredetected with the aid of the detection probe. As is the case with thecapture probe, it is also possible to correspondingly protect thedetection probe against enzymatic extension/break-down and to add itbefore or during amplification.

In a preferred embodiment, the nucleic acid to be detected is amplifiedin the presence of a capture probe immobilized to a solid phase whiledetectable, labelled nucleotides, especially mononucleotides, areenzymatically incorporated. After a possible separation of excessmononucleotides the nucleic acid immobilized to the solid phase isdetermined via the amount of incorporated detectable nucleotides. Aparticularly preferred amplification for the invention is the methoddescribed in EP-A-0 329 822. It is a particular advantage of the methodof this specification to modify the capture such that the RNA(transcripts) to be immobilized are protected against degradation by thehybrid of capture probe and RNA.

When an immobilizable capture probe is used, the invention proposes twoembodiments. If a first embodiment, the nucleic acid to be detected isamplified in the presence of the immobilizable capture probe anddetectably labelled mononucleotides. All known amplification methods canbe employed. A preferred method is the polymerase chain reaction (EP-B-0201 184) with thermal cycles allowing denaturing between the cycles; theamplification reaction can be carried out in any desired vessel.Subsequently, the reaction mixture is transferred into a vessel to thesolid phase of which the immobilizable capture probe can be immobilized.It is preferred to separate excess mononucleotides and to use the labelat the solid phase as a measure for the presence of the nucleic acid tobe detected.

In a second, preferred embodiment, the amplification reaction alreadyoccurs in a vessel where the immobilizable nucleic acid can be bound tothe inner wall. It is an advantage that a transfer of the amplificationmixture into another vessel is no longer required. If thermocyclicallycontrolled amplification reactions are used, e.g. PCR or LCR, theinvention proposes to use thermostable vessel having correspondingcoatings.

Provided the reaction vessel is already suitable for detecting hybridsand when direct labels are used, the reaction can be controlled from thestart of the amplification until the end of the detection without addingreagents.

Currently known methods have the disadvantage that after theamplification procedure, the reaction mixture had to be exposed to theenvironment to add the probes (contamination). If the probes are addedtogether with the amplification reagents, the procedure advantageouslyrequires fewer pipetting steps and, secondly, the reaction vessel mustnot be opened again after amplification and addition of reagents (e.g.capture probe). This characteristic feature is particularly advantageousfor methods with direct labels which do not require the addition ofreagents to detect the label. In such cases, e.g.electrochemiluminescence labels, no reagent is added from the start ofthe amplification until the measurement of the analyte-dependent signal.The addition of regents is, however, possible at any time.

Another subject matter of the invention are nucleic acids which aremodified to be protected against both enzymatic break-down of nucleicacids in hybrids of the nucleic acid and complementary nucleic acids aswell as enzymatic extension and, further, their use as capture probes toimmobilize nucleic acids. In a preferred manner, these nucleic acids aremodified at positions of the nucleobase, especially at the functionalamino groups, which are capable of establishing hydrogen bridges tocomplementary nucleic acids. They are not modified with respect to thenatural nucleobases.

Another subject matter of the invention is a reagent kit comprising afirst container A with an enzyme for extending nucleic acids and asecond container B with a capture probe in accordance with theinvention.

This or other containers preferably contain all other reagents necessaryfor immobilization and amplification or detection of nucleic acids. Suchreagents are buffers, mononucleoside triphosphates, labelling reagentsand the like.

A particularly preferred container B features either a detectabledetection probe which is also modified in accordance with the inventionor detectable mononucleoside triphosphates. Moreover, container A cancontain a nucleic acid especially an enzyme which degrades RNA in aRNA/DNA-hybrid.

Subject matter are also special electrochemiluminescence-labelledmononucleoside triphosphates.

Groups which have electrochemiluminescent properties are known. They arehereinafter referred to as electrochemiluminescene group E. Particularlypreferred electrochemiluminescent components of E are coordinationcomplexes K with an atomic weight of more than 500 g/mol, preferably 550to 2000 g/mol. Preferred metal ions are ions of the VIIb and VIIIbsubgroups of the periodic table, particularly Ru, Os, Re, with rutheniumbeing particularly preferred.

Suitable ligands of the metal ion in the coordination complex are inparticular organic ligands or groups as described in WO 92/10267 orEP-A-0 340 605.

Suitable organic ligands are hydrocarbons which contains atoms whichacts as electron donors for the corresponding metal atom, e.g. nitrogen,oxygen, or sulfur. The electron donors are suitably disposed in ageometric arrangement so as to allow complex formation with the metalion. In case of ruthenium, the bipyridil residues are preferred ligands.

Moreover, via one of the ligands, the complex is bound to amononucleoside triphosphate. In a preferred manner, a spacer group A isdisposed between the coordination complex and the atoms of thenucleoside triphosphate. The spacer group preferably is an atomic chainconsisting of 3, particularly 5 to 15, more particularly 4 to 10 atomswith additional groups being bound to the side of the chain, ifnecessary. The chain of atoms preferably contains a hydrocarbon chainwhich can be interrupted by heteroatoms, e.g. --O--(oxygen) or an NR²-group where N is nitrogen and R² hydrogen or a C₁ -C₆ alkyl group. Thechain of atoms preferably contains once the component --(CR³ ₂ --CR³ ₂--O) wherein R³ is hydrogen or a C₁ -C₆ alkyl group. The spacer grouphas a preferred molecular weight of less than 1000 g/mol.

Preferably, the electrochemiluminescene group E is covalently bound toan atom of the nucleobase N of the mononucleotide.

Preferred sites for the linking are C5, C6, C4 or N4 for pyrimidines andC8 or N6 for purines and C7 for deazapurines.

Preferred mononucleoside triphosphates are those of formula I

    P--Z--B--E                                                 (I)

wherein

P is a triphosphate group or a triphosphate analog group

Z is a sugar or sugar analog group

B is a nucleobase or nucleobase analog group and

E is an electrochemiluminescene group.

Triphosphate, sugar, and nucleobase are the groups contained innaturally occurring mononucleoside triphosphates. Analog groups arethose groups which are obtained by replacing one or several atoms, butyet do not essentially inhibit the property of the compounds of formulaI, namely the incorporation in nucleic acids.

Triphosphate analogs are groups, for example, where one or severaloxygen atoms are replaced by sulfur atoms. Sugar analogs are groupswhere the endocyclic oxygen atom is replaced by the --CR⁴ ₂ --group,wherein R⁴ is hydrogen or a C₁ -C₆ alkyl group. Nucleobase analogs are,for example, deazapurines, preferably 7-deazapurines.

Preferred mononucleotide triphosphates are compounds of the formula Ia##STR1## wherein X is hydrogen or the OR¹ -group

R¹ is a C₁ -C₆ alkylene group of hydrogen

B is a purine, deazapurine or a pyrimidine group

A is a spacer group

and

K is an electrochemiluminescent coordination complex.

and the alkali and earth alkali salts thereof.

Experience has surprisingly shown that even groups as large as thedescribed metal complexes do not essentially interfere with theincorporation of mononucleoside triphosphates labelled therewith bymeans of enzymes catalyzing the incorporation of mononucleosidetriphosphates in nucleic acids. The incorporated amount of labelledmononucleoside triphosphates is sufficient for the detection of nucleicacid labelled therewith by means of electrochemiluminescence. The methodof the invention exhibits a higher degree of sensitivity in comparisonto prior art methods using electrochemiluminescene.

It is particularly surprising to see the very high dynamic measurementrange, i.e. the ratio of the greatest quantifiable amount of nucleicacids to be determined at a given concentration of mononucleosidetriphosphates to the lowest quantifiable amount. This ratio can exceedvalues of 10⁷.

The mononucleoside triphosphates can be incorporated in different ways.

One way is the extension of oligonucleotides, so called primers, whichare hybridized to the nucleic acid to be detected. A segment of anucleic acid is attached to the 3'-end of the primer in a reaction thatis catalyzed by a polymerase, preferably DNA-polymerase. This segment isessentially complementary to the corresponding segment of the nucleicacid to be detected. This reaction can occur cyclically using the formedextension products while amplifying a segment of the nucleic acidpresent. Such a method is described in EP-A-0 201 184.

Another possibility is the replication or transcription of nucleic acidswhich contain an origin of replication or a promotor. In this case, thenucleic acids are composed solely of mononucleotides without primerextension. Examples are known from WO 91/03573 or WO 91/02818.

During the incorporation, at least one type of mononucleotides ((d)ATP,(d)CTP, (d)GTP, or dTTP/(d)UTP) is partially or completely replaced inthe reaction mixture by the electrochemiluminescent mononucleosidetriphosphates. In a preferred manner 10 to 50% of one type ofmononucleoside triphosphates (e.g. dUTP) are replaced by the labelledanalog (e.g. ruthenium-complex-labelled dUTP). A preferred concentrationof the labelled mononucleoside triphosphate ranges between 20 and 70 μM,particularly preferred is a range between 25 and 40 μM. All otherconditions for the incorporation reaction do not essentially differ fromthose mentioned in the above mentioned documents.

Depending on their structure, the electrochemiluminescene-labelledmononucleoside triphosphates can be produced in different manners.

If the group is to be attached to the nucleobase, an amino group of thenatural bases can be reacted with an electrophilic group, e.g. anactivated ester group of the spacer group.

It has proven to be particularly expedient to react those mononucleosidetriphosphates with such a reactive electrochemiluminescent compoundwhich already possesses at one atom of the base a spacer group with anamino group. An example are the mononucleoside triphosphates containingsubstituted in position 5 with an alkyl amino group. (Proc. Acad. Sci.USA 78, 6633-37 (1991).

An electrochemiluminescent compound having a reactive group can bepurchased, for example, from the IGEN company. The amide is formed underconditions similar to those applying to the regular formation of amidesfrom activated esters and amines.

FIG. 1 is a diagram showing the detection method in accordance with theinvention using PCR for the amplification.

FIG. 2 is a diagram showing the detection method in accordance with theinvention using NASBA for the amplification.

FIG. 3 is a graph showing the results of HpBadw21-DNA determinations inaccordance with the invention (PCR with probe) as compared to PCRwithout probe (dark bars).

FIG. 4 is a similar graph for Listeria determinations using NASBA (withprobe; dark bars).

FIG. 5 shows the result of an HIV 1 determination usingelectrochemiluminescent labels in the presence of the capture probe withNASBA.

FIG. 6 is a reaction scheme for the amplification and detection ofnucleic acids using NASBA where a digoxigenin-labelled mononucleoside isincorporated in the transcripts.

FIG. 7 shows the structure of Ru(bpyr)₃ -AA-dUTP.

FIG. 8 shows the structure of Ru(bpyr)₃ -DADOO-dUTP.

FIG. 9 is picture of an agarose gel showing the differences in themolecular weight of the labelled nucleic acid.

FIG. 10 shows the detection of amplification products with differentamounts of either (bpyr)₃ AA dUTP or Ru(bpyr)₃ -DADOO-dUTP incorporatedby hybridization with a Biotin-labelled probe in the gel.

FIG. 11 is a graph showing the dependencies of the signal height uponthe amount of ruthenium-labelled mononucleotides used in the reaction.

FIG. 12 is another graph showing the quantity-dependent detection ofnucleic acid when ruthenium labels are incorporated.

FIG. 13 is a graph comparing the different embodiments of the invention.The bar with the vertical lines stands for an embodiment where a secondvessel was used to specifically bind the capture probe whereas the barswith the cross lines stand for an example where amplification and wallbinding were carried out in one single vessel. The dotted bars relate toan embodiment where the reaction mixture for detecting the color insolution which developed during the enzymatic reaction is transferred ina detection vessel.

Each of the references and patent documents cited in the presentapplication are hereby incorporated by reference into the presentapplication. Specifically, these documents include EP-A-0 079 139;EP-A-0 192 168; WO 92/06216; PCT/US92/01735; WO 91/15499; EP-A-0 324474; EP-A-0 329 822; EP-B-0 201 184; WO 92/10267; EP-A-0 340 605; WO91/03573; WO 91/02818; EP-A-0 200 362; EP-A-0 269 092; WO 90/08197;EP-B-0331 127; Schmitz, G. G., et al. (1990) Anal. Biochem. 192,222;Inoue, H., et al. (1987) FEBS Lett. 215, 327-330; Iribarren, A. M., etal., (1990) PNAS 87, 7747-7751; Maniatis et al., Molecular Cloning: ALaboratory Manual, CSH, 194-195, 1982; Kievits, T. et al. (1991), J. ofVirol Methods, 273-286; Leland et al., (1990), J. Electrochem. Soc.,137, 3127-33; Langer et al., (1981) Proc. Natl. Acad SCI, USA, 78,6633-37, 1981; Bergstrom et al. (1977), J. Carbohydr., Nucleosides,Nucleotides, 4:257; Ono et al. (1983), Nucl. Acids Res. 11:1747-1757;Kessler et al. (1980) "Nonradioactive labeling and Detection of Nucleicacids", Biol. Chem. Hoppe-Seyler 371:917-927; and Blackburn et al.(1991) Clin. Chem. 37:1534-1539.

The following examples are intended to further illustrate the invention:

EXAMPLE 1 Detection of HBV with Direct and Subsequent Use of a CaptureProbe

Hpbadw21 in a concentration of 1 ng to 100 atg per reation mixture wereused as a target for the polymerase chain reaction (PCR)(EP-A-0 200 362)The volume for the PCR mixture was 100 μl.

The mixture was composed as follows:

    ______________________________________                                        Final concentration                                                           ______________________________________                                        Primer 1 (SEQ. ID. NO. 1) 200 nM                                                                         Primer 2 (SEQ. ID. NO. 2) 200 nM                     dATP (Boehringer Mannheim) 200 μM                                          dCTP (Boehringer Mannheim) 200 μM                                          dGTP (Boehringer Mannheim) 200 μM                                          dTTP (Boehringer Mannheim) 175 μM                                          dig-11-dUTP (Boehringer Mannheim GmbH, 25 μM                               Best. Nr 1093088)                                                             PCR-Puffer 1 X                                                                Taq-DNA-Polymerase (Perkin Elmer) 2.5 U                                       PCR-Mix 99 μl                                                              Sample 1 μl                                                                Volume 100 μl                                                            ______________________________________                                         10 X PCR Puffer: 100 mM Tris/HCl, 500 mM KCl, 15 mM MgCl.sub.2, 1 mg/ml       gelatine, pH 9.0                                                              Primer 1: Oligodeoxynucleotide, 18 mer, d (GGAGTGTGGATTCGCACT) (Pos.          2267-2284; EMBL, subtype adw, SEQ. ID. NO. 1)                                 Primer 2: Oligodeoxynucleotide, 18 mer, d (TGAGATCTTCTGCGACGC) (Pos.          2436c-2419c, EMBL, Subtype adw, SEQ. ID. NO. 2)                               Hpbadw 21: cloned HBVDNA; nucleotide 29-2606 (EMBL) of the                    HBV.sub.adwsequence cloned in pUCBM20 (Boehringer Mannheim) and               linearized.                                                                   Biotinylated capture probe: d (AGCCTATAGACCACCAAATGCCCCTAT) 5biotinylated     (SEQ. ID. NO. 3) Pos. 2290-2316; EMBL, subtype adw Ref.: Ono et al.           (1983). Nucl. Acids Res. 11                                              

The PCR mixtures were amplified in a Perkin Elmer Thermal Cycler underthe following cycle conditions: 3'93° C.; (1'94° C., 1'50° C., 2'70°C.)×30; 5'95° C., 37° C.

The biotin-labelled probe was in a concentration of 100 ng per reactionmixture. During the PCR, the probe was block at its 3'-end withBio-16-ddUTP (Boehringer Mannheim, Cat. No. 1427, 598) Ref.: Schmitz, G.G. et al. (1990) Anal biochem. 192, 222).

Streptavidin-coated microtiterplates were used for the detection(Boehringer Mannheim GmbH, EP-A-0 269 092).

1. Detection of PCR without probe followed by hybridization:

Following the amplification procedure, 40 ng of biotin-labelled captureprobe were added to 20 μl of a PCR reaction mixture. This mixture washeat up to 95° C. for 5', then cooled on ice and for 15 min incubated at37° C. Then, 180 μl hybridization buffer were added (50 mM phosphatebuffer, 750 mM NaCl, 75 mM Na citrate, 0.05% BSA, pH 6.8); this mixturewas then pipetted into the well of a streptavidin-coated (SA)microtiterplate and for 1 hour incubated at 37° C. After washing themixture 3 times with 0.9% NaCl, 200 μl of conjugate buffer 100 mMTRIS×HCl, 0.9% NaCl, 1% BSA, pH 7.5 with 0.2 U/ml anti-digoxigenin-POD(Boehringer Mannheim) were added by pipetting and for 20 min incubatedat 37° C. Once again washed 3 times, 200 μl ABTS® (1.9 mmol/l) were usedfor the detection and the absorbance was measured at Hg 405 nm.

2. Detection of the PCR with the probe being used directly during thePCR (as proposed by the invention)

The amplification was carried out in the presence of 100 ng of captureprobe.

Following the amplification 180 μl hybridization buffer were addeddirectly to 20 μl of a PCR reaction mixture and treated as describedunder 1.

The results are shown in FIG. 3.

EXAMPLE 2 Promotor-Controlled Amplification With and Without Direct Useof a Probe (NASBA)

The analyte used was isolated chromosomal Listeria monocytogenes DNA(see WO 90/08197) in concentrations of 50 ng to 5 fg per reactionmixture. NASBA is described in EP-A-0 329 822.

The NASBA is preceded by a reaction to convert the double-strandedanalyte DNA into single-stranded DNA using the T₇ -RNA polymerasepromotor sequence (PreNASBA). This is normally achieved by usingSequenase®. Principally, however, any DNA polymerase is suitable.

    ______________________________________                                                             Final concentration in the                                 1 PreNASBA-(Prereaction) PreNASBA                                           ______________________________________                                        10 X NASBA-Buffer    1 X                                                        Primer 3 0.2 μM                                                            dATP (Pharmacia) 1 mM                                                         dCTP (Pharmacia) 1 mM                                                         dGTP (Pharmacia) 1 mM                                                         dTTP (Pharmacia) 1 mM                                                         ATP (Pharmacia) 2 mM                                                          CTP (Pharmacia) 2 mM                                                          GTP (Pharmacia) 2 mM                                                          UTP (Pharmacia) 2 mM                                                          dig11-UTP (Boehringer Mannheim) 10-20 μM                                   Best. Nr. 1209256)                                                            +Target DNA                                                                   PreNASBA-Mix 20 μl                                                       ______________________________________                                    

This mixture was heated up to 95° C. for 5 minutes and then cooled onice.

It was adjusted to 10 mM dithiothreitol (DTT); then, 13 U T₇ -DNApolymerase (Sequenase®, by United States Biochemicals) were added. Themixture was then incubated for 15 min at 37° C. and again heated up to95° C. and again cooled on ice.

11A NABSA Amplification: Reaction Procedure 1

a. Composition of reaction mixture

The volume used for the NASBA was 25 μl (23 μl NASBA-Mix+2 μlPreNASBA-Mix, from example 2, Part 1).

    ______________________________________                                                               Final concentration                                      Reagent in NASBA                                                            ______________________________________                                        10 X NASBA-Buffer      1 X                                                      DTT 10 mM                                                                     dATP 1 mM                                                                     dCTP 1 mM                                                                     dGTP 1 mM                                                                     dTTP 1 mM                                                                     ATP 2 mM                                                                      CTP 2 mM                                                                      GTP 2 mM                                                                      UTP 2 mM                                                                      dig UTP 10-20 μM                                                           RNase Inhibitor (by Boehringer Mannheim) 0.48 U                               T.sub.7 -RNA-Polymerase (by Boehringer Mannheim) 80 U                         AMV-RT (by Boehringer Mannheim) 4 U                                           RNaseH (by Boehringer Mannheim) 1 U                                           Primer 3 0.2 μM                                                            Primer 4 0.2 μM                                                            DMSO 15%                                                                       23 μl                                                                     +Product from PreNASBA 2 μl                                                 25 μl                                                                   ______________________________________                                         10 x NASBA Puffer: 400 nM TRIS/HCl, 500 mM KCl, 120 mM MgCl.sub.2, pH 8.5     Primer 3: d (AATTCTAATACGACTCACTATAGGGAGACGCGCTTTA CCTGCTTCGGCGATT), SEQ.     ID. NO. 4 PIIIRegion (Pos. 35-56). The underlined portion is the              T.sub.7RNA-Polymerase Promotor sequence.                                      Primer 4: d (GTAATCATCCGAAACCGCTCA), SEQ. ID. NO. 5 PIIIRegion (Pos.          196c-216c)                                                               

The NASBA amplification was allowed to occur for 1.5-2 hours at 40° C.

b. Hybridization

After the amplification, 200 ng of biotinylated probe were added to theNASBA mixture and heated up to 95° C. for 5 min and then cooled on ice.Then, the mixture was incubated for 30 min at 37° C.

Probe: d(CGTTTTACTTCTTGGACCG). SEQ ID. No. 6 at the 5'-end biotinylatedwith biotinamite (Applied Biosystems) PIII region (Pos. 162c-180c)

c. Detection in microtiterplates

195 μl hybridization buffer (50 mM phosphate buffer, 750 mM NaCl, 75 mMNa-citrate, 0.05% BSA, pH 6.8) were added to 5 μl of the hyridizationmixture. The mixture was then pipetted into the well of amicrotiterplate and incubated for another hour at 37° C. After washingthree times with 0.9% NaCl solution, incubation was continued for 20minutes with 200 μl 0.2 U/ml anti-digoxigenin-POD (Boehringer Mannheim)in conjugate buffer (100 mM TRIS-HCl, 0.9 NaCl, 1% BSA, pH 7.5) at 37°C. The mixture was washed again three times and then 200 μl ABTS®(Boehringer Mannheim) (1.9 mmol/l) were added and the absorbance wasmeasured at Hg 405 nm.

11B NASBA Amplification: Reaction Procedure 2

a. Composition of reaction mixture

The composition of the NASBA reaction mixture corresponded to the oneused in 11A. In addition, the capture probe was added directly duringamplification in a concentration 140 ng per mixture.

A 2'-allylribooligonucleotide which was biotin-labelled (biotin amide byApplied Biosystems Inc.) at the 5'-end and blocked with a hydrophiliclinker at its 3'-end was used for the direct employment of the captureprobe during the NASBA reaction. A 2'-O-allyl-modifiedoligoribonucleotide was used as a probe as this "RNA strand" is notdegraded by RNAseH in RNA/DNA hybrids (Ref.: Inoue, H. et al (1987) FEBSLett. 215, 327-330). The 3'-end of the oligonucleotide was blocked toavoid a non-specific extension during the polymerase reaction.

sequence: 5'-biotin-CGU UUU ACU UCU UGG ACC -G-3'-block

(SEQ ID. No. 7)

C=2'--O--allyl cytidine

G=2'--O--allyl guinosine

A=2'--O--allyl adenosine

U=2'--O---allyl uridine ##STR2##

This capture probe was synthesized with 2'--O--allyl nucleosidephosphorus amidites by Boehringer Mannheim Biochemicals according to theprotocol by Iribarren et al. (Ref.: Iribarren, A. M. et al. (1990) PNAS87, 7747-7751) using an oligonucleotide synthesizer by Pharmacia (GeneAssembler)

b. Hybridization

does not apply

c. MTP detection

The procedure corresponded to the one used for Ic with 5 ml of NASBAmixture.

The results are summarized in FIG. 4.

EXAMPLE 3 Detection of HIV-1 Using NASBA

In the following example, HIV-1 was detected with the aid of the NASBAamplification system (EP-A-0 329 822). NASBA was carried out in thepresence of the biotin-labelled capture probe. The amplification productwas detected directly via the incorporated label (ruthenium-labelledUTP).

The tests covered HIV-positive sera of patients with AIDS or ARC (AIDSrelated complex). The RNA was isolated after the Guanidinum Hot PhenolMethod according to Maniatis et al. [Molecular Cloning: A laboratorymanual (Cold Spring Harbor Lab., Cold Spring Harbor, N.Y. 194-195, 1982)and the RNA solution were stored at -70° C. in aliquots of 10 μl. Priorto the NASBA reactions, the RNA solution were diluted 1:10, 1:100,1:1000, 1:10000 and 2 μl were used in the NASBA reaction.

For the NASBA reaction and the composition of the reaction mixture,refer to example 2 IIb: 30 μM ruthenium-labelled UTP (=Ru(bpyr)₃-AA-UTP) were used instead of digUTP (for details on the synthesis seeexample 4).

Primers and probe used:

    __________________________________________________________________________    OT 188:                                                                           AATTCTAATACGACTCACTATAGGGCCTGGCTTTAATTTTACTGGTA                              P1 (SEQ. ID. NO. 8)                                                          OT 42: ACAGGAGCAGATGATACAGTATTAG                                               P2 (SEQ. ID. NO. 9)                                                          OT 15: 5'-Bio-UGGAAGAAAUCUGUUGACUCAGAUUGGUUGC-block-3'                         Sample (SEQ. ID. NO. 10)                                                   __________________________________________________________________________

The underlined portion of sequence OT 188 corresponds to the T₇-RNA-polymerase promotor.

Oligonucleotide OT 15 is again a 2'-O--allyl ribooligonucleotide blockedat its 3'-end (see example 2 IIB) and biotinylated at its 5'-end.

Ref.: Kievits, T. et al. (1991). J. of Virol. Methods 273-286.

Detection of the Amplification Products

After the reaction, 10 μg (2×10⁶ beads) magnetic beads (Dynabeads®M-280. Streptavidin, Dynal) in 20 μl phosphate buffer (62 mMNa-phosphate, 0.94 M NaCl, 94 mM Na-citrate, pH 7) were added to 5 μl ofNASBA reaction mixture and incubated for 30 minutes at 37° C.Subsequently, a magnet was used to collect the beads at the bottom ofthe sample cup. The were washed twice each time with 100 μl phosphatebuffer. Then, the beads were suspended in 100 μl of said phosphatebuffer and 250 μl ECL assay buffer (0.2 M KH₂ PO₄, 0.05 M NaCl, 0.1 Mtripropylamin, pH 7.5; Ref.: J. K. Leland et al. (1990). J. Electrochem.Soc. 137:3127-33) were added. The measurement was carried out on an ECLAnalyser (Origen 1.0; Manufactured by Igen Inc.) with a photomultipliervoltage of 940 V. The measurement time was 2 seconds at a ramp of 2 Vper second. The entire measuring cycle for each sample was 2 minutes.

The result is summarized in FIG. 5.

EXAMPLE 4 Synthesis of Ru(bpyr)₃ -AA-UTP

Origen® label (IgenInc.)=[4-(N-succinimidyloxycarbonylpropyl)-4'-methyl-2,2'-bipyridine-bis(2,2'-bipyridine)]-ruthenium(II)-dihexaflurophosphate

AA-UTP=5-(3-aminoallyl)-uridine triphosphate was prepared according toLanger et al. [Proc. Natl. Acad. Sci. USA. 78, 6633-37, 1981]

DMSO=dimethylsulfoxide

5.5 mg (0.01 mmol) AA-UTP (lithium salt) were dissolved in 2 ml 0.1 Msodium borate buffer (pH 8.5). To achieve this, a solution of 15.8 mg(0.015 mmol) of the Origen® label were added to 1.5 ml DMSO and stirredovernight at room temperature. Subsequently, the Ru(bpyr)₃ -AA-UTPproduct was purified by means of ion exchange chromatography (SephadexDEA, Cl form; gradient; 0--0, 4 M LiCl). Yield: 7 mg (46% oftheoretical).

EXAMPLE 5 Synthesis of Ru(bpyr)₃ -AA-dUTP and Ru(bpyr)₃ -DADOO-dUTP

5-(3-aminoallyl)-dUTP (=AAdUTP) was prepared according to Langer et al.(1981). Proc. Natl. Acad. Sci. USA. 78:6633-37; DADOO-dUTP was obtainedby reacting 5-mercury-dUTP withN-allyl-N'-(8-amino-3,5-dioxa-octyl)urea.

Synthesis of Ru(bpyr)₃ -AA-dUTP (FIG. 7)

3.2 mg AAdUTP were dissolved in 0.1 M sodium borate buffer and stirredovernight at RT with a solution of 5.6 mg of the Origen® label in 560 μlDMSO.

The reaction was controlled via TLC and paper electrophoresis and theproduct was then purified by means of ion exchange chromatography.

Yield: 3.8 mg (48% of theoretical)

[M+H]⁺ : 1180.1

Synthesis of Ru(bpyr)₃ -DADOO-dUTP (FIG. 8)

a. Synthesis of N-allyl-N'-(8-amino-3,5-dioxaoctyl)urea

15 g (0.1 mol) diaminodioxaoctane (Merck) were cooled to -40° C. in 500ml diisopropylether. 5.5 g (0.06 mol) allylisocyanate (Fluka) were addedslowly and dropwise. After stirring for 30 min. at -40° C., the mixturewas allowed to reach RT and the diisopropylether was removed bydistillation. The remainder was stirred for 1 h with 600 ml aceticester, the precipitate was removed by filtration and washed three timeswith acetic ester. The combined acetic ester fractions were concentratedand the resulting product was purified by means of flash chromatographywith an acetic ester/methanol solvent gradient of 9:1 to 8:2.

Yield: 4.5 g (29% of theoretical) colorless liquid.

b. Reaction with 5-mercury-dUTP

5-mercury-dUTP (=HgdUTP) was prepared according to Bergstrom et al.(1977). J. Carbohydr., Nucleosides, Nucleotides 4:257.

370 mg (0.54 mmol) HgdUTP and 113 mg K₂ PdCl₄ are dissolved in 20 mlsodium acetate buffer (pH 5). Then, 0.5 g (2.16 mmol)N-allyl-N'-(8-amino-3,5-dioxaoctyl)urea were added dropwise and stirredovernight at RT. The precipitate is removed by filtration, the filtrateis concentrated and purified by means of ion exchange chromatography(DEAE-Sephadex-CL-Form) with a buffer gradient of 0 to 0.3 M LiCl.Subsequently, the product is precipitated twice form aceton/ethanol=2:1;the precipitate is removed by filtration, washed and dried. The resultis 60 mg DADOO-dUTP (15.7% of theoretical).

c. The synthesis was carried out as described for Ru(bpyr)3-AA-dUTP byreacting DADOO-dUTP with the Origen® label.

EXAMPLE 6 PCR Amplification and Labelling of HBV-DNA with Ru(bpyr)₃-AA-dUTP and Ru(bpyr)₃ -DADOO-dUTP

Portions of 1 ng Hpbadw21 were amplified; the volume of the PCR reactionmixture was 100 μl (80 μl PCR Mix, 20 μl sample).

The PCR Mix had the following composition:

    ______________________________________                                        Reagent        Concentration of                                                                          Final concentration                                  or sample stock-solution in the PCR                                         ______________________________________                                        Hpbadw211                  ng/100 μl                                         Primer 1 5 μM 200 nM                                                       Primer 2 5 μM 200 nM                                                       dATP (Li-Salt) 4 mM 200 μM                                                 dCTP (Li-Salt) 4 mM 200 μM                                                 dGTP (Li-Salt) 4 mM 200 μM                                                 dTTP (Li-Salt) 4 mM 133 μM-200 μM                                       Ru(bpyr)3-AA-dUTP 700 μM 66 μM-0.01 μM                               or                                                                            RU(bpyr)3-DADOO-dUTP 300 μM 66 μM--0.01 μM                           PCR-Puffer 10 x 1 x                                                           Taq-Polymerase 5 U/μl 2.5 U                                              ______________________________________                                         10 x PCRPuffer: 100 mM Tris/HCl, 500 mM KCl, 15 mM MgCl.sub.2, 100 mg/ml      gelatine, pH 9.0                                                              Primer 1: oligodeoxynucleotide, 18 mer, d (GGAGTGTGGATTCGCACT) SEQ. ID.       NO. 1 (Pos. 2267-2284; EMBL. subtype adw)                                     Primer 2: oligodeoxynucleotide, 18 mer d (TGAGATCTTCTGCGACGC) SEQ. ID. NO     2 (Pos. 2436c-2419c; EMBL; subtype adw)                                       Hpbadw 21: adw 21 cloned in pUC BM 20 (Boehringer Mannheim) and linearize

The PCR reaction mixtures were amplified in a Perkin Elmer ThermalCycler with 30 cycles of 60 sec at 94° C., 60 sec at 50° C., and 120 secat 70° C.

The Ru(bpyr)₃ -AA-dUTP and Ru(bpyr)₃ -DADOO-dUTP concentrations variedbetween 66 μM, 30 μM, 10 μM, 1 μM, and 0.1 μM to 0.01 μM, the dTTPconcentrations were increased correspondingly to have a total Ru(bpyr)₃-dUTP/dTTP concentration of 200 μM in the amplification mixture.

To have a comparison, the same PCR reaction mixture was tested wheredifferent concentrations of digoxigenin-11-dUTP (Boehringer Mannheim,Cat. No. 1039 088) were incorporated and also a PCR reaction mixturewhere no modified nucleotides were incorporated.

    __________________________________________________________________________          Ru(bpyr)3    Ru(bpyr)3                                                      dUTP   dUTP                                                                  dTTP (short)  dTTP (long)  dTTP DigdUTP  DTTP                                No. μM μM No. μM μM No. μM μM No. μM                   __________________________________________________________________________      1 133 66 1/1 133 66 1/2 133 66 1/3 133                                        2 170 30 2/1 170 30 2/2 170 30 2/3 170                                        3 190 10 3/1 190 10 3/2 190 10 3/3 190                                        4 199 1 4/1 199 1 4/2 199 1 4/3 200                                           5 200 0.1 5/1 200 0.1 5/2 200 0.1                                             6 200 0.01 6/1 200 0.01 6/2 200 0.01                                        __________________________________________________________________________    Lane Mixture No.                                                                             Lane                                                                              Mixture No.                                                                             Lane                                                                              Mixture No.                                  __________________________________________________________________________      I 1      IX 3           XVI 4/3                                               II 1/1         X 3/1                XVII 5                                    III 1/2        XI 3/2               XVIII 5/1                                 IV 1/3         XII 3/3              IXX 5/2                                   V 2      XIII 4         XX 6                                                  VI 2/1         XIV 4/1              XXI 6/1                                   VII 2/2        XV 4/2               XXII 6/2                                  VIII 2/3                                                                    __________________________________________________________________________

The different PCR reaction mixtures were applied onto a 1% agarose gelin portions of 10 μl and the amplification products were made visible byethidium bromide (FIG. 9). Owing to its higher molecular weight, theamplification product with incorporated Ru(bpyr)₃ -DADOO-dUTP (=longspacer) is more retarded on the gel than the Ru(bpyr)₃ -AA-dUTP product(=short spacer). This, however, occurs only when higher concentrationsof modified nucleotide are present and thus a correspondingly largeramount of label is incorporated during the amplification reaction. Theresult is an increase in the molecular weight as compared to theunmodified product.

EXAMPLE 7 Filter Hybridization with a Biotinylated Oligonucleotide Probe

Biotinylated capture probe:

d(AGA CCA CCA AAT GCC CCT AT) SEQ. ID. NO. 11 biotinylated withbiotinamidite (Applied Biosystems) corresponds to Pos. 2297-2316 of theHBV_(adw) -sequence; EMBL sequence data bank. Ref: Ono et al. (1983).Nucl. Acids Res. 11:1747-1757.

10 μl of the PCR reaction mixtures 1-6, 1/1-6/1 and 1/3-2/3 were appliedonto a 1% agarose gel, then blotted onto a nylon membrane and hybridizedat 45° C. overnight with portions of 100 ng of the above describedbiotinylated oligonucleotide probe corresponding to C. H. Kessler et al.(1980) "Non-radioactive labelling and detection of nucleic acids", Biol.Chem. Hoppe-Seyler 371:917-927. Biotin was detected with a conjugateconsisting of streptavidin and alkaline phosphatase (BoehringerMannheim, Cat. No. 1093 266), NBT/X-phosphate (Boehringer Mannheim, Cat.No. 1383 213 and 760 986). As shown in FIG. 10, the desiredamplification product could be detected in all PCR reaction mixtures bymeans of hybridization using an oligonucleotide probe specific for theamplification product.

EXAMPLE 8 Hybridization and Detection

8.1 "Two-step assay"

10 μl of the mixtures 1, 4, and 6 as well as 1/1, 4/1, and 6/1 werediluted 1:10 with 0.05 M NaOH. After 5 min, 100 μl of this mixture werethen neutralized with 500 μl of hybridization buffer 62.5 mMNa-phosphate, 0.94 M NaCl, 94 mM Na-citrate, pH=6.5) which alreadycontained the biotinylated oligonucleotide probe in a concentration of94 ng/ml. At the same time, the PCR reaction mixtures 1, 4, and 6 aswell as 1/1, 4/1 and 6/1 were subjected to thermal denaturing by heatingthem up to 100° C., and subsequent cooling on ice. After hybridizing for1 hour at 375° C., 50 μl of a suspension of streptavidin-coated magneticparticles (Dynabeads® M-280, streptavidin, Dynal) in hybridizationbuffer (concentration 600 μg/ml) were added and incubated for anotherhour at 37° C. Using a magnet, the magnetic beads were then collected onthe bottom of the sample cup and the supernatant solution was decantedand the beads were again washed twice, each time with 500 μl phosphatebuffer (50 mM Na-phosphate buffer; pH=7.4). Finally, the beads weresuspended in 200 μl phosphate buffer, then 500 μl ECL assay buffer wereadded (see Blackburn et al. (1991). Clin. Chem. 37:1534-1539 andmeasured on an ECL analyzer (Origen® 1.0, IGEN, Rockville, USA).

The measurement was carried out with a photomultiplier voltage of 940 V.The measurement time was 2 sec. at a ramp voltage of 2 V per second. Thecomplete measuring cycle for each sample was 2 minutes.

8.2 "One-step assay"

In this second and more simple assay procedure, denaturing of the PCRreaction mixture and hybridization with the biotinylated capture probewere carried out as described for the two-step-assay. After 2 hours ofhybridization at 37° C. another 50 μl of magnetic bead were pipettedinto this solution and then directly measured in the ECL analyzerwithout "bound/free" separation. The blank measured was thecorresponding concentration of Ru(bpyr)₃ -AA-dUTP in the hybridizationbuffer.

The voltage of photomultiplier was 800 V, all other measurementconditions corresponded to those described above.

FIG. 11 also shows the ECL peak intensities measured in dependency ondifferent Ru(bpyr)₃ -AA-dUTP and Ru(bpyr)₃ -DADOO-dUTP concentrations inthe amplification mixture.

EXAMPLE 9 Detection of HBV Plasmid-DNA

The target was again Hpbadw 21. It was used with the followingconcentrations: 1 ng, 1 pg, 100 fg, 1 fg, 200 ag. Amplification wascarried out corresponding to the conditions of example 2: once using 10μM (Ru(bpyr)₃ -AA-dUTP/190 μl dTTP and once using 30 μM Ru(bpyr)₃-AAdUTP/170 μl dTTP final concentration in the PCR. Followinghybridization with the biotinylated capture probe (Seq. Id. No. 3) andbinding to SA magnetic beads according to the "one-step-assay" without"bound/free" separation, the amplification product was measured in theOrigen® 1.0 Analyzer. The photomultiplier voltage used was 900 V, allother conditions corresponded to those described under item 4.2.

FIG. 12 shows the measured ECL peak intensities in dependency on thedifferent target concentrations.

EXAMPLE 10 Detection of HBV Using the Probe Directly During Thre PCR inMicrotiterplates Coated with Thermostable SA

The PCR mixture was prepared as described in Example 1. However, thevolume of the mixture was reduced to 50 μl and covered with 30 μl Sigmamineral oil (Sigma Mineral Oil, light white oil M-3516. The primers, thebiotinylated capture probe II, and the template as well as theconcentrations used were identical. The PCR was carried out in Techneplates (Cat. No. 140800) coated with thermostable streptavidin (SA)prior to starting the procedure. A MW-2 Techne-Cycler was used for theamplification. The cycle conditions were the same as those used inexample 1. After the PCR, the mixture was allowed to stand for 30 min at37° C. Then it was washed three times (see example 1) and the plate wasincubated with 100 μl conjugate buffer (see Example 1) and the plate wasincubated with 100 μl conjugate buffer (see Example 1) for each well.Subsequently it was washed again three times and then reacted with 100ml ABTS® solution (see Example 1). The absorbance measured is shown inFIG. 13. The bars on the right side (SA-MTP PCR) show the absorbancesfor the various concentrations.

EXAMPLE 11 Detection of HBV Using the Probe Directly During Thre PCR inMicrotiterplates Coated with Thermostable SA

The vessels used were 0.5 ml Perkin Elmer cups (Cat. No.: N801.0537 forthe PCR Cycler 9600. The insides of the cups were coated withthermostable streptavidin according to EP-B-0 331 127. The reactionmixture was prepared as in Example 10. However, the volume of thereaction mixture was 100 μl and the PCR was not covered with oil.Conjugate buffer and ABTS® were added to the cups in portions of 200μl/cup. After incubating with ABTS® for 15 min, the mixture wastransferred into a Nunc plate (Cat. No.: 838713) and measured in thereader. The absorbances measured are given in FIG. 13 (center bars).

EXAMPLE 12 Coating the Techne Plates and the EP-Cups with ThermostableStreptavidin

The coating was carried out according to EP-B-0 331 127.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 11                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                          - - GGAGTGTGGA TTCGCACT             - #                  - #                      - #  18                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                          - - TGAGATCTTC TGCGACGC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                          - - AGCCTATAGA CCACCAAATG CCCCTAT          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 52 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                          - - AATTCTAATA CGACTCACTA TAGGGAGACG CGCTTTACCT GCTTCGGCGA TT - #                 52                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                          - - GTAATCATCC GAAACCGCTC A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                          - - CGTTTTACTT CTTGGACCG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: RNA                                               - -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                          - - CGUUUUACUU CUUGGACCG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                          - - AATTCTAATA CGACTCACTA TAGGGCCTGG CTTTAATTTT ACTGGTA   - #                    47                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                          - - ACAGGAGCAG ATGATACAGT ATTAG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: RNA                                               - -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                         - - UGGAAGAAAU CUGUUGACUC AGAUUGGUUG C        - #                  - #              31                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                         - - AGACCACCAA ATGCCCCTAT            - #                  - #                      - # 20                                                                 __________________________________________________________________________

We claim:
 1. A mononucleoside triphosphate having the formula I:

    P-Z-B-E                                                    (I)

wherein P is a triphosphate group or a triphosphate analog group, Z is asugar or sugar analog group, B is a nucleobase analog group, and E is anelectrochemiluminescence group.
 2. The triphosphate according to claim1, wherein said triphosphate has the structural formula Ia: ##STR3## oralkali and earth alkali salts thereof, whereinX is hydrogen or OR¹, R¹is selected from the group consisting of C₁ -C₆ alkyl group, a C₁ -C₆alkylene group and hydrogen, B is selected from the group consisting ofa purine, deazapurine and a pyrimidine group, A is a spacer group, and Kis an electrochemiluminescent coordination complex.
 3. The triphosphateaccording to claim 1, wherein the electrochemiluminescence group is acoordination complex of a metal ion and at least one organic ligandhaving an atomic weight greater than 500 g/mol.
 4. The triphosphateaccording to claim 1, wherein the molecular weight of E is between 550and 2000 g/mol.
 5. A method for the extension of an oligonucleotidecomprising the steps of:hybridizing an oligonucleotide to a firstnucleic acid, and extending said oligonucleotide by a mononucleosidetriphosphate having the formula I

    P-Z-B-A-K                                                  (I)

at the 3'-end of said oligonucleotide using a polymerase, wherein P is atriphosphate group or a triphosphate analog group, Z is a sugar or asugar analog group, B is a nucleobase or a nucleobase analog group, andK is an electrochemiluminescent coordination complex, and A is a spacergroup,wherein the molecular weight of K is between 550 and 2000 g/mol.6. The method according to claim 5, wherein said triphosphate has thestructural formula Ia: ##STR4## or alkali and earth salts thereof,whereinX is hydrogen or OR¹, R¹ is selected from the group consisting ofa C₁ -C₆ alkyl group, a C₁ -C₆ alkylene group and hydrogen, B isselected from the group consisting of a purine, deazapurine and apyrimidine group, A is a spacer group, and K is anelectrochemiluminescent coordination complex.
 7. A method for thedetermination of a nucleic acid comprising the steps ofhybridizing aprimer to the nucleic acid to be determined, extending the 3'-end ofsaid primer by attaching a mononucleoside triphosphate having theformula I:

    P-Z-B-A-K                                                  (I)

wherein P is a triphosphate group or a triphosphate analog group, Z is asugar or a sugar analog group, B is a nucleobase or a nucleobase analoggroup, K is an electrochemiluminescent coordination complex, and A is aspacer group,using a polymerase, to produce an extension product anddetermining any electrochemiluminescence group in the extension productas a measure of the nucleic acid to be determined.
 8. The methodaccording to claim 7, wherein the extension reaction is performed in acyclic manner.
 9. The method according to claim 7, wherein saidtriphosphate has the structural formula Ia: ##STR5## or alkali and earthsalts thereof, whereinX is hydrogen or OR¹, R¹ is selected from thegroup consisting of a C₁ -C₆ alkyl group, a C₁ -C₆ alkylene group andhydrogen, B is selected from the group consisting of a purine,deazapurine and a pyrimidine group, A is a spacer group, and K is anelectrochemiluminescent coordination complex.